Neoinnervation and neovascularization of acellular pericardial-derived scaffolds in myocardial infarcts

Engineered bioimplants for cardiac repair require functional vascularization and innervation for proper integration with the surrounding myocardium. The aim of this work was to study nerve sprouting and neovascularization in an acellular pericardial-derived scaffold used as a myocardial bioimplant. To this end, 17 swine were submitted to a myocardial infarction followed by implantation of a decellularized human pericardial-derived scaffold. After 30 days, animals were sacrificed and hearts were analyzed with hematoxylin/eosin and Masson's and Gallego's modified trichrome staining. Immunohistochemistry was carried out to detect nerve fibers within the cardiac bioimplant by using βIII tubulin and S100 labeling. Isolectin B4, smooth muscle actin, CD31, von Willebrand factor, cardiac troponin I, and elastin antibodies were used to study scaffold vascularization. Transmission electron microscopy was performed to confirm the presence of vascular and nervous ultrastructures. Left ventricular ejection fraction (LVEF), cardiac output (CO), stroke volume, end-diastolic volume, end-systolic volume, end-diastolic wall mass, and infarct size were assessed by using magnetic resonance imaging (MRI). Newly formed nerve fibers composed of several amyelinated axons as the afferent nerve endings of the heart were identified by immunohistochemistry. Additionally, neovessel formation occurred spontaneously as small and large isolectin B4-positive blood vessels within the scaffold. In summary, this study demonstrates for the first time the neoformation of vessels and nerves in cell-free cardiac scaffolds applied over infarcted tissue. Moreover, MRI analysis showed a significant improvement in LVEF (P = 0.03) and CO (P = 0.01) and a 43 % decrease in infarct size (P = 0.007)

Unravelling the effects of mechanical physiological conditioning on cardiac adipose tissue-derived progenitor cells in vitro and in silico

Mechanical conditioning is incompletely characterized for stimulating therapeutic cells within the physiological range. We sought to unravel the mechanism of action underlying mechanical conditioning of adipose tissue-derived progenitor cells (ATDPCs), both in vitro and in silico. Cardiac ATDPCs, grown on 3 different patterned surfaces, were mechanically stretched for 7 days at 1 Hz. A custom-designed, magnet-based, mechanical stimulator device was developed to apply ~10% mechanical stretching to monolayer cell cultures. Gene and protein analyses were performed for each cell type and condition. Cell supernatants were also collected to analyze secreted proteins and construct an artificial neural network. Gene and protein modulations were different for each surface pattern. After mechanostimulation, cardiac ATDPCs increased the expression of structural genes and there was a rising trend on cardiac transcription factors. Finally, secretome analyses revealed upregulation of proteins associated with both myocardial infarction and cardiac regeneration, such as regulators of the immune response, angiogenesis or cell adhesion. To conclude, mechanical conditioning of cardiac ATDPCs enhanced the expression of early and late cardiac genes in vitro. Additionally, in silico analyses of secreted proteins showed that mechanical stimulation of cardiac ATDPCs was highly associated with myocardial infarction and repair.Peer ReviewedPostprint (published version

A Cell-Enriched Engineered Myocardial Graft Limits Infarct Size and Improves Cardiac Function

Altres ajuts: Fundació La Marató de TV3 [201502; 201516]; Beca de Recerca Bàsica de l'Acadèmia de Ciències Mèdiques i de la Salut de Catalunya i de Balears 2015; Beca d'Investigació Bàsica de la Societat Catalana de Cardiologia 2015; Generalitat de Catalunya (SGR 2014); Sociedad Española de Cardiología; Fundació Privada Daniel Bravo AndreuMyocardial infarction (MI) remains a dreadful disease around the world, causing irreversible sequelae that shorten life expectancy and reduce quality of life despite current treatment. Here, the authors engineered a cell-enriched myocardial graft, composed of a decellularized myocardial matrix refilled with adipose tissue-derived progenitor cells (EMG-ATDPC). Once applied over the infarcted area in the swine MI model, the EMG-ATDPC improved cardiac function, reduced infarct size, attenuated fibrosis progression, and promoted neovascularization of the ischemic myocardium. The beneficial effects exerted by the EMG-ATDPC and the absence of identified adverse side effects should facilitate its clinical translation as a novel MI therapy in humans

The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload

BackgroundPulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) A2 receptor (TP) antagonist NTP42 attenuates experimental PAH across key hemodynamic parameters in the lungs and heart. This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction.MethodsThe effects of NTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM).ResultsIn the MCT-PAH model, NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM.ConclusionThis study shows that, through antagonism of TP signaling, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions

Physiological conditioning of adult progenitor cells boosts cardiac regeneration = El condicionament fisiològic de cèl·lules progenitores adultes promou la regeneració cardíaca

[eng] The optimal cell lineage for cardiac regeneration remains elusive; and current treatments, with the exception of heart transplantation, are not enough to restore myocardial function. Thus, new strategies, such as cardiac tissue engineering, are now emerging as a new therapeutic modality. On the other hand, cardiac cells are normally subjected to electrical and mechanical forces that regulate gene expression and cellular function. Therefore, our hypothesis claims that in vitro individual or combined synchronous electro-mechanical stimuli mimicking the cardiac environment, could mature or induce cardiac differentiation on therapeutic cells to benefit further retention and integration into the myocardium. This thesis proposal had several goals: 1. Design an ad-hoc device to supply electrical and mechanical stimuli, individually or synchronously, to a monolayer cell culture. 2. Design a protocol for electrical, mechanical and electromechanical conditioning. 3. Analyze the cardiomyogenic effect of electrical, mechanical and electromechanical conditioning on adipose tissue-derived progenitor cells (ATDPCs) and cardiomyocyte progenitor cells (CMPCs). 4. Study the feasibility of a tissue engineered construct with trained cardiac adipose tissue-derived progenitor cells (cardiac ATDPCs). 5. Examine the regenerative capacity of electromechanically conditioned cardiac ATDPCs within a myocardial infarction murine model. Summing up, the conclusions derived from this thesis project are: 1. A new device for ad-hoc electrical and mechanical stimulation, individually or synchronously, has been developed in collaboration with the Electronic and Biomedical Instrumentation Group from the Universitat Politècnica de Barcelona. This device has been patented (WO 2013185818 A1) including some results obtained in this thesis. 2. Protocols for electrical, mechanical and electromechanical conditioning on cardiac ATDPCs culture were designed and optimized with the aim to mimic the cardiac environment. 3. Electrical stimulation on cardiac ATDPCs and CMPCs enhances the expression of cardiac transcription factors (MEF2A and GATA-4) crucial for cardiac differentiation. In addition, electrical conditioning promotes cardiac ATDPCs elongation and cell alignment. 4. Mechanical stimulation on cardiac ATDPCs upregulates cardiac transcription factors (GATA-4 and Tbx5) and structural genes (α-actinin and cTnI) expression, strongly dependent on the patterned surface. 5. In vitro electro-mechanical stimulation pre-commits the cell population against the hostile cardiac milieu through increased expression of cardiac transcription factors (Tbx5 and GATA-4), structural genes (b-MyHC) and, most importantly, calcium handling genes (Cx43 and SERCA2). 6. Conditioned cardiac ATDPCs fibrin constructs scarcely migrate to the murine myocardium, de novo express cTnI in vivo, increase vessel density and promote the sprouting of functional blood vessels, and improve cardiac function with only 105 implanted cells.[cat] El llinatge cel·lular òptim per a la regeneració cardíaca continua sent un gran desconegut. Els tractaments actuals són insuficients per a restablir la funció cardíaca, exceptuant el trasplantament de cor. Per això, noves estratègies, com l’enginyeria tissular cardíaca, estan emergent com a noves modalitats terapèutiques. D’altra banda, les cèl·lules cardíaques estan normalment subjectes a estímuls elèctrics i mecànics, que regulen la seva expressió gènica i la funció cel·lular. Per tant, la hipòtesi d’aquest treball és que el pre-condicionament in vitro, mitjançant estímuls biofísics similars a l’entorn cardíac, podria madurar i induir cert grau de diferenciaicó cardíaca a les cèl·lules terapèutiques. Així es milloraria la seva retenció i integració al miocardi hoste, i beneficiaria el tractament de l’infart de miocardi. Els objectius d’aquest treball són: 1. Disseny i producció d’un aparell apte per a portar a terme el condicionament elèctric i mecànica, individualment o de manera combinada, a un cultiu cel·lular en monocapa de cèl·lules progenitores derivades de teixit adipós cardíac (ATDPCs cardíaques). 2. Disseny d’un protocol per al condicionament elèctric, mecànic i electromecànic. 3. Caracterització de les cèl·lules obtingudes de cadascun dels condicionaments. 4. Estudi de la viabilitat d’un constructe d’enginyeria amb ATDPCs cardíaques condicionades. 5. Examinar l’efecte del condicionament electromecànic de les ATDPCs cardíaques implantades, mitjançant un constructe tridimensional, en el model murí d’infart de miocardi. Les conclusions obtingudes són: 1. S’ha desenvolupat un aparell apte per a l’estimulació elèctrica i mecànica, individual o sincrònicament, en col·laboració amb el Grup d’Instrumentació Electrònica i Biomèdica de la Universitat Politècnica de Catalunya. Aquest aparell s’ha patentat (WO 2013185818 A1) amb alguns dels resultats derivats d’aquesta tesi. 2. S’ha dissenyat i optimitzat un protocol per al condicionament elèctric, mecànic i electromecànic de les ATDPCs cardíaques amb la intenció de mimetitzar l’ambient cardíac. 3. L’estimulació elèctrica de les ATDPCs cardíaques i les CMPCs millora l’expressió de factors de transcripció (MEF2A i GATA-4) crucials per a la diferenciació cardíaca. A més, el condicionament elèctric de les ATDPCs cardíaques promou la seva elongació i alineament acord al patró de la superfície. 4. L’estimulació mecànica de les ATDPCs cardíaques incrementa l’expressió genètica de factors de transcripció (GATA-4 i Tbx5) i gens estructurals (α-actinin i cTnI), i és fortament dependent del patró de la superfície. 5. L’estimulació electromecànica in vitro prepara les ATDPCs cardíaques per a l’hostil entorn cardíac augmentant l’expressió de factors de transcripció cardíacs (Tbx5 i GATA-4), gens estructurals (β-MyHC) i gens relacionats amb el maneig del calci (Cx43 i SERCA2). 6. El pegat de fibrina amb ATDPCs cardíaques electromecànicament entrenades promou una escassa migració al miocardi murí, una lleu expressió proteica de cTnI, l’increment de la densitat vascular a la vora de la cicatriu de l’infart, la formació de vasos sanguinis funcionals al miocardi i al constructe, i una millora de la funció cardíaca del 8% amb només 105 cèl·lules

Adaptation and Maladaptation of the Right Ventricle in Pulmonary Vascular Diseases

The right ventricle is coupled to the low-pressure pulmonary circulation. In pulmonary vascular diseases, right ventricular (RV) adaptation is key to maintain ventriculoarterial coupling. RV hypertrophy is the first adaptation to diminish RV wall tension, increase contractility, and protect cardiac output. Unfortunately, RV hypertrophy cannot be sustained and progresses toward a maladaptive phenotype, characterized by dilation and ventriculoarterial uncoupling. The mechanisms behind the transition from RV adaptation to RV maladaptation and right heart failure are unraveled. Therefore, in this article, we explain the main traits of each phenotype, and how some early beneficial adaptations become prejudicial in the long-term

The battle of new biomarkers for right heart failure in pulmonary hypertension: is the queen of hearts NT-proBNP defeated at last?

Therapeutic cell differentiatio

Author Correction: Unravelling the effects of mechanical physiological conditioning on cardiac adipose tissue-derived progenitor cells in vitro and in silico

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper

Post-infarction scar coverage using a pericardial-derived vascular adipose flap. Pre-clinical results

BACKGROUND: Myocardial salvage after coverage with a fat flap was recently demonstrated in acute coronary occlusion. The effect of this novel therapeutic strategy on a chronic myocardial scar is unknown. METHODS: Myocardial infarction (MI) was induced by coil deployment in the mid circumflex artery in the swine model. Two weeks after infarction, a pericardial-derived adipose flap was transposed, fully covering the scar, in the treated group. Infarct size and histopathology were analyzed on post mortem sections. To assess cell migration, adenoviral eGFP vectors were injected in the adipose flap and expression was evaluated upon sacrifice both at the flap and myocardium. Magnetic resonance imaging (MRI) was used to measure left ventricular (LV) ejection fraction and ventricular volumes at baseline, 2 weeks post-MI, and at 6 weeks. RESULTS: One month after flap transposition, histopathology confirmed a 34% reduction in infarct size (8.7% vs. 5.7%; P=0.04) and the presence of vascular connections at the flap-myocardium interface. High eGFP expression was detected at the infarct core both at the gene and protein level (negligible signal was detected at the flap on sacrifice). At the functional level, changes in LV ejection fraction and volumes (end-systolic and end-diastolic) were not significantly different between groups (all P values>0.1). CONCLUSIONS: Our data support the use of post-infarction scar coverage with a pericardial-derived fat flap to reduce infarct size, due partly to neovascular connections and cell trafficking at the flap-myocardium interface. Further studies are needed to validate the functional and clinical relevance of this intervention